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Anti-folate receptor alpha antibody, its conjugate and use thereof

An anti-folate and conjugate technology, applied in the field of biomedicine, can solve the problems of low side effects, high specificity of monoclonal antibody therapeutic target, limited curative effect, etc. Effect

Active Publication Date: 2022-07-08
烟台迈其生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Monoclonal antibody therapy has attracted more and more attention due to its high target specificity and low side effects, but its curative effect is relatively limited when used alone

Method used

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  • Anti-folate receptor alpha antibody, its conjugate and use thereof
  • Anti-folate receptor alpha antibody, its conjugate and use thereof
  • Anti-folate receptor alpha antibody, its conjugate and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Preparation of murine anti-FRα monoclonal antibody

[0053] Monoclonal antibodies were prepared by immunizing mice with the extracellular domain protein FOLR1 (abcam) of FRα as an antigen. 0.15ml of FOLR1 protein (75μg protein dissolved in PBS buffer) and 0.15ml of Freund's complete adjuvant (Sigma) were mixed evenly in equal volumes, and Balb / c mice were taken and injected subcutaneously in the back.

[0054] A total of 5 mice were immunized, and each mouse was injected with 0.3 ml. The second injection was performed after a 2-week interval. The amount of antigen injected into each mouse was the same as the first injection. The third injection was performed after a 4-week interval. The amount of antigen injected into each mouse was the same as the first injection. Blood drawn.

[0055] The serum of the mice was detected by enzyme-linked immunosorbent assay (ELISA), and the spleen of the mouse with the highest anti-FOLR1 antibody titer in the serum was taken...

Embodiment 2

[0059] Example 2 Humanization of anti-FRα monoclonal antibodies

[0060] Humanization was performed by grafting the light and heavy chain CDRs of a murine anti-FRα monoclonal antibody into human IgG1 variable regions.

[0061] The heavy chain variable region (VH) of a humanized anti-FRα monoclonal antibody was designed, and an amino acid residue His in the framework region 3 (FWR3) of the human germline heavy chain was converted into a murine isomeric residue Arg, The conformation of the CDRs is preserved in the reconstituted FOLR1-binding antibody.

[0062] At the same time, the light chain variable region (VL) of the humanized anti-FRα monoclonal antibody was designed, and the amino acid residues Val and Tyr in the human germline light chain FWR2 were converted into the mouse isomeric residues Ser and Ser, and the The conversion of amino acid residues Ala and Tyr in the human germline light chain FWR3 to the murine isopositional residues Thr and Phe preserves the conformati...

Embodiment 3

[0069] Example 3 Preparation of Antibody Conjugates

[0070] Prepare reducing buffer: dissolve TCEP (Tris-2-carboxyethyl-phosphine, tris(2-carboxyethyl) phosphine) and DTPA (diethylenetriaminepentaacetic acid, diethylenetriaminepentaacetic acid) in PBS, both substances in reducing buffer The concentrations were 0.26 mM and 2 mM, respectively.

[0071] Antibody reduction: 20 mg / mL mAb (in PBS buffer) was mixed with reducing buffer at a volume ratio of 1:1, and the reaction was stirred at 25° C. for 2 h.

[0072] Preparation of small molecule drug solution: The small molecule toxins MC-VC-PAB-MMAE, MC-VC-PAB-MMAF or MC-MMAF were dissolved in DMSO (dimethyl sulfoxide, dimethyl sulfoxide), respectively, to a final concentration of 10 mM.

[0073] Coupling: add 25% DMSO to the reduced protein, then slowly add the small molecule drug solution according to the molar ratio of the small molecule drug to the antibody 4.4 for coupling, and stir for 1 h at 25°C. Finally, the conjugated ...

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Abstract

The invention discloses an anti-folate receptor alpha antibody, a conjugate thereof and uses thereof, belonging to the technical field of biomedicine. The antibody includes a heavy chain and a light chain, the heavy chain comprises three CDR regions, and the three CDR regions of the heavy chain have amino acids as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively Sequence; the light chain comprises three CDR regions, and the three CDR regions of the light chain have the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively. The antibody has a strong affinity for FRα; the three ADCs after the naked antibody is coupled to the therapeutic agent through a linker have similar affinities to FRα as the naked antibody. The naked antibody and the three ADCs of the present invention can be used to target and inhibit the proliferation of FRα-positive tumor cells, and then be used to prepare medicines for the treatment or prevention of choriocarcinoma, ovarian cancer and lung cancer.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, in particular to an anti-folate receptor alpha antibody, its conjugate and use thereof. Background technique [0002] Folate receptor α (FRα) is a glycosylphosphatidyl alcohol-coupled glycoprotein with high affinity for folic acid and its derivatives, and transports folic acid into cells through endocytosis. FRα is encoded by the FOLR1 gene and consists of 7 exons and 6 introns with a length of 6 800 kb. FRα is not expressed or the expression level is very low in normal tissues, but it is highly expressed in malignant tumors such as ovarian cancer, fallopian tube cancer, endometrial cancer, choriocarcinoma, primary peritoneal cancer, lung cancer, breast cancer and other malignant tumors of specific epithelial origin. Express. [0003] Monoclonal antibody therapy has received more and more attention due to its high target specificity and low side effects, but its efficacy is limited ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28A61K47/68A61P35/00
CPCC07K16/28A61K47/6889A61K47/6811A61K47/6817A61P35/00C07K2317/24C07K2317/56C07K2317/565C07K2317/92
Inventor 姚雪英李乐涵
Owner 烟台迈其生物科技有限公司
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